| As one of the most widely used herbicides in the world,the toxic mechanism of glyphosate to mammals has been the focus of researchers.Evidence has shown that glyphosate has toxic effects on mammalian tissues and organs,such as the heart,liver,and kidney.Glyphosate affects the physiological functions of tissues and organs by causing apoptosis,impairing DNA replication,and other mechanisms,and has potential carcinogenic effects.In addition,some studies have also found that glyphosate,as a low-toxicity chemical,may have cytotoxic,genotoxic or carcinogenic effects on mammals.Glyphosate also acts as an estrogen analogue that disrupts the endocrine system of mammals,thereby affecting the reproductive and metabolic functions of the body.As an important metabolic organ in mammals,the liver has physiological functions such as hormone secretion,detoxification,and maintenance of glucose and lipid metabolism homeostasis.Long-term ingestion of glyphosate can lead to steatosis in animal livers,causing non-alcoholic fatty liver disease,and at the same time causing disturbances in the body’s glucose metabolism.The above evidence suggests that glyphosate ingestion impairs glucose and lipid metabolism in mammalian livers.However,the specific mechanism of glyphosate on hepatic glucose and lipid metabolism is still unclear and needs to be further investigated.The circadian clock is an intrinsic regulatory mechanism in mammals,which can regulate the rhythmic expression of a variety of glucose and lipid metabolism-related genes and play an important role in maintaining the homeostasis of mammalian glucose and lipid metabolism.A previous study of our group found that the inhibitory effect of glyphosate on mammalian testosterone was mediated through the circadian clock signaling pathway.Accordingly,we hypothesized that the effect of glyphosate on hepatic glucose and lipid metabolism in mammals may have a potential connection with the circadian clock signaling pathways.In this study,from the perspective of circadian clock,we established a glyphosate exposure model on mouse AML12 hepatocytes and wild-type male mice of the ICR strain,and used quantitative real-time PCR(q PCR),western blotting(WB),periodic acid-schiff(PAS)staining,kit quantitative detection and other experimental techniques,to explore the mechanism of glyphosate affecting hepatic glucose and lipid metabolism in mouse liver.The results of the study are as follows:1.CCK-8 kit and flow cytometry were used to screen out the maximum glyphosate treatment concentration(0.1 m M)that had no significant effect on the viability and apoptosis of AML12 cells,and this concentration was used to treat AML12 cells after forskolin synchronization.q PCR and WB were used to detect the effects of glyphosate on the rhythm and expression levels of circadian clock genes and glucose and lipid metabolism related genes in AML12 cells.The results showed that the m RNA expressions of circadian clock genes Bmal1,Nr1d1 and Dbp in AML12 cells in the control group and glyphosate-treated group had rhythmic changes.Glyphosate at 0.1 m M significantly inhibited the m RNA expressions of circadian clock genes Bmal1,Nr1d1 and Dbp in AML12 cells,and decreased the expression of NR1D1 protein.At the same time,0.1 m M glyphosate significantly inhibited the m RNA expression of Fasn,Pparα,Hmgcr and Glut2 and other glucose and lipid metabolism related genes in AML12 cells.2.In order to explore the effects of glyphosate on the circadian clock system and glucose and lipid metabolism in mice,we reviewed the literature and finally determined that the model was established by freely drinking 0.5% glyphosate aqueous solution for 8 weeks.The control group mice drink glyphosate-free distilled water freely.During the modeling period,the behavioral activity rhythm,body weight,food intake and water intake of the mice were accurately measured.After successful modeling,blood and liver tissue samples were collected from mice.The effects of glyphosate on the hepatic circadian clock system and glucose and lipid metabolism in mice were detected by various techniques such as glucose tolerance test,insulin tolerance test,PAS staining,PCR,WB and kit quantitative detection.The results showed that compared with the control group,the activity of the mice in the glyphosate-treated group increased significantly under light conditions during the modeling period,and there was no significant difference in feed intake,but water intake decreased significantly;weight gain,although not significantly different from the control group,showed a decreased trend.The results of glucose tolerance test showed that the rate of glucose absorption and metabolism in the glyphosate-treated mice was significantly lower than that in the control group.The results of insulin tolerance test showed that the insulin sensitivity and blood glucose raising ability of glyphosate-treated mice were significantly lower than those of control mice.The results of PAS staining and quantitative detection of the kit showed that the liver glycogen content of the glyphosate-treated mice was significantly lower than that of the control mice.The quantitative detection results of the cholesterol detection kit showed that the liver cholesterol content of the mice in the glyphosate-treated group was significantly lower than that of the mice in the control group.The results of q PCR and WB indicated that the m RNA expressions of the liver circadian clock genes Bmal1,Nr1d1 and Dbp in the control and glyphosate-treated mice were rhythmic,but compared with the control group,the phases of the circadian clock genes of the mice in the glyphosate-treated group had different degrees of phase delay.There was no significant difference in the overall m RNA expression of the above circadian clock genes.In addition,compared with the control group,the liver BAML1 protein of the glyphosate-treated mice showed a downward trend,but the difference was not significant,and the overall expression of NR1D1 protein had no significant difference compared with the control group.Compared with the control group,the overall m RNA expression of hepatic glucose metabolism-related genes Glut2,Cd36 and Pdk4 in the glyphosate-treated mice decreased significantly.At the same time,the m RNA expression of hepatic lipid synthesisrelated genes Srebp-1c and Fasn was significantly higher than that in the control group at zeitgeber time(ZT)20,and the Pparγ gene was significantly lower than the control group at ZT4,the m RNA expression of lipid decomposition-related gene Pparα was significantly higher than that in the control group at the ZT20 time point.As revealed by the above evidence,glyphosate treatment inhibited the expression of some clock genes and glucose and lipid metabolism related genes in AML12 cells,disrupted the activity rhythm of mice,and reduced the accumulation of glycogen and cholesterol in mice liver,resulting in the disorder of glucose and lipid metabolism in mice.However,there are still some limitations in this study.The current results cannot prove that the liver circadian clock genes are involved in the effects of glyphosate on hepatic glucose and lipid metabolism,and there is still a lack of in-depth analysis of the mechanism of glyphosate affecting liver glucose and lipid metabolism.In conclusion,this study provides a preliminary basis for indepth exploration of the specific mechanism of glyphosate on mammalian circadian clock and glucose and lipid metabolism,and provides theoretical support for evaluating the liver toxicity of glyphosate. |
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